Structured illumination imaging was performed using a Zeiss Elyra 7 lattice SIM equipped with a ×63 C-Apochromat ×63/1.20 W (Zeiss) water immersion objective, four excitation lasers (405 nm diode, 488 nm OPSL, 561 nm OPSL, and 642 nm diode laser) and two sCMOS (PCO, pco.edge 4.2) cameras. Fourfold expanded gels were transferred onto PDL coated single well chamber slides (CellVis, #C1-1.5H-N). For excitation of DAPI the 405 nm laser diode was set to 10% output power, for excitation of Dextran-Alexa488 the 488 nm laser to 20%. A respective exposure time of 100 and 200 ms was set for the two channels. Z-stacks were captured in 3D Leap mode with optimal step intervals and appropriate filter sets (BP 420–480 + LP 655 and BP 495–550 + BP 570–620 for the DAPI- and 488-channel, respectively). SIM imaging was performed by capturing 13 phase shifts of the lattice SIM pattern per slice. Super-resolved images are processed in ZEN 3.0 SR FP2 (black) (Zeiss, Version 16.0.10.306) with SIM and SIM² processing modules.
Elyra 7 lattice sim
Manufactured by Zeiss
Sourced in Germany
The Elyra 7 Lattice SIM is a high-resolution imaging system developed by Zeiss. It utilizes Lattice Structured Illumination Microscopy (Lattice SIM) technology to achieve super-resolution imaging capabilities. The core function of the Elyra 7 Lattice SIM is to provide researchers with the ability to capture detailed and high-quality images of microscopic samples.
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4 protocols using elyra 7 lattice sim
1
Structured Illumination Microscopy of Extracellular Matrix
2
Confocal and Lattice SIM Imaging
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For the confocal imaging, Zeiss LSM510, LSM700 or LSM800 microscopes were used with a 63×1.4 NA oil objective. Zeiss Elyra 7 Lattice SIM was used to visualize the fine detail of tagged actin incorporation presented in Fig. 5D,E . ZEN (2009, 5.5 & 3.0 black for Elyra), ImageJ (1.54f), and Imaris (9.8.2) were used to process the images.
3
Apoptosis Pathway Profiling Using Annexin V-mCherry
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Following H/R, the cell culture medium was aspirated into a suitable centrifuge tube and the cells were rinsed once with PBS. An appropriate volume of trypsin cell digestion solution was added for cell detachment. The cells were collected, transferred to a centrifuge tube, and centrifuged at 2,000 rpm for 5 min, The supernatant was discarded, and the cells were gently suspended in PBS. The resuspended cells were centrifuged at 2,000 rpm for 5 min, and the supernatant was discarded. In total, 194µL Annexin V-mCherry Binding Buffer was added to gently resuspend the cells. Furthermore, 5 µL Annexin V-mCherry and 1µL GreenNuc™ Caspase-3 Substrate (1 mM) were added and mixed gently. The mixture was incubated at room temperature (20–25°C) for 20–30 min in the dark. GreenNuc™-DNA exhibited green fluorescence (excitation/emission=500/530 nm), whereas Annexin V-mCherry displayed red fluorescence (excitation/emission=587/610 nm). Cells were collected and gently suspended in 100 µL Annexin V-mCherry Binding Buffer. After smearing, the cells were observed using an Elyra 7 Lattice SIM (Zeiss, Germany). We followed identical procedures for flow cytometry experiments, employing Gallios (Beckman Coulter) flow cytometers for the analyses, and the data were analyzed using CytExpert software packages.
4
Fluorescent Imaging of Photoautotrophic Cells
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For imaging of fluorescently tagged lines, photoautotrophically grown cells were immobilized on 1.5% (w/v) low-melting point agarose in TP medium. Indirect immunofluorescence of RBCS2-APEX2 was performed according to Uniacke et al. (2011) (link) with the following modifications: cells were fixed with 3.7% (w/v) formaldehyde solution in PBS for 30 min at room temperature. Anti-Flag antibody (F1804; Sigma-Aldrich) at 1:1,000 dilution in PBS containing 1% (w/v) BSA was used as primary antibody. Anti-Mouse Alexa Fluor plus 555 (A32727; Invitrogen) was used as the secondary antibody at 1:1,000 dilution. Labeled cells were then kept in the dark prior to imaging. Images were taken using a Zeiss LSM880 microscope with the Airyscan module or a Zeiss Elyra7 Lattice SIM. Excitation and emission filters of fluorophore and chlorophyll autofluorescence were set as follows: mVenus (excitation: 514 nm; emission: 520 to 550 nm); chlorophyll (excitation: 633; emission: 610 to 650 nm); and mCherry/mScarlet-I/Alexa Fluor plus 555 (excitation: 561 nm; emission: 580 to 600 nm).
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